The ability to manipulate large DNA fragments is very important to contemporary biology. Although recombinant techniques were developed more than 25 years ago, improved methodologies remain highly prized, especially to aid functional genomic studies (1, 2). Conventional approaches for the manipulation of DNA are multi-step and time-consuming, involving the digestion of DNA by appropriate restriction enzymes followed by ligation into a suitable cloning vector. A critical factor in the success of traditional DNA manipulation is the length of the DNA to be engineered. Due to the demands of restriction specificity it is very difficult to manipulate DNA molecules of more than 20,000 base pairs using traditional restriction-ligation methods (3). This is of particular concern to bacterial artificial chromosome (BAC) engineering, where there is often a need to engineer DNA fragments large enough to contain the appropriate regulatory elements for a certain gene's expression (4). BACs have become powerful tools in functional genomic studies (1), so better methods for precisely manipulating BAC DNA are required.
Recently, scientists have developed a new method, known as recombineering, to manipulate large DNA fragment (5-10), and have overcome the difficulties in traditional BAC DNA engineering. These methods have been based either on the RecET proteins or the λ-recombination proteins. The RecET system is based on homologous recombination mediated by the RecE and RecT proteins. The Red system is based on homologous recombination mediated by three λ-recombination proteins, exo (α), bet (β) and gam (γ), collectively known as the Red proteins. The Red system is similar to the RecET system, but has been shown to be 50-100 times more efficient in Escherichia coli (6). A modified DH10B strain, called DY380, harboring a defective λ-prophage carrying the red genes under the tight control of the temperature sensitive λ-cI857 repressor has been created (10). Incubation of DY380 cells at 42° C. results in the inactivation of the temperature-sensitive λ repressor, and hence the production of the exo (α), bet (β) and gam (γ) proteins, enabling recombination. In the present study, a two-step approach based on the Red-mediated recombination was developed. In the first step, retrieval, a large DNA fragment is retrieved from a BACs. In the second step, recombination, the large DNA fragment is inserted into a second BAC to form the unified BAC containing the entire desired sequence. This task would be extremely difficult by conventional means.